A 4.4 kilobase segment of Bacillus subtilis DNA has been cloned that contains two genes whose transcription appears to be under the control of spore development. These genes specify discrete-sized RNAs of 0.4 and 0.3 kilobases and are referred to as the 0.4 kb and the 0.3 kb gene respectively. The 0.4 kb gene is turned on at an early stage of sporulation (within the first hour) and, as evidence that it is under sporulation control, its transcription is blocked in six different mutants arrested at the stage 0 of spore formation. The 0.3 kb gene is first activated at an intermediate stage of development (during the third hour) and its transcription appears to be blocked in both stage 0 and stage II mutants. In addition to these two sporulation-controlled genes, the cloned B. subtilis DNA contains a "vegetative" gene that is actively transcribed during both growth and sporulation. The location and direction of transcription of these three genes has been determined by hybridization to separated endonuclease restriction fragments and hybridization to separated DNA strands. The cloned B. subtilis DNA has now been transferred from the original cloning vector pMB9, an E. coli plasmid, to pUB110, a plasmid that replicates in B. subtilis. This has permitted propagation of the B. subtilis DNA in its homologous host and may provide a handle on genetic analysis of its sporulation-controlled genes. In vitro studies of the transcription of the cloned 4.4 kilobase DNA have been initiated. Purified RNA polymerase holoenzyme from vegetative cells actively transcribes the vegetative gene in vitro but does not copy RNA from the two sporulation-controlled cistrons. A recently detected activity in crude extracts of sporulating cells selectively and asymmetrically transcribes the 0.4 kb gene.